ABSTRACT V&#945;14 invariant natural killer T (V&#945;14iNKT) cell is a unique subset of thymic-derived innate T cells that express T cell receptor (TCR) and NK cell markers. Unlike conventional T cells, V&#945;14iNKT cells respond to glycolipid antigen presented by CD1d expressed on antigen presenting cells (APCs). Several studies have reported important protective roles for activated V&#945;14iNKT cells during viral infections by replication-competent viruses. V&#945;14iNKT cell activation during viral infection occurs through host lipids presented by CD1d or by a TCRindependent mechanism involving Toll-like receptors (TLRs). TLRs are pathogen recognition receptors that play a critical role in initiating the innate immune response to microbes. Among the TLRs, TLR7, TLR8 and TLR9 sense viral signals and can stimulate APCs to induce V&#945;14iNKT cell activation during viral infections by replication-competent viruses. The potential role of TLR3, also a viral signal sensor, in mediating V&#945;14iNKT cell activation during acute viral infections are not known, and is therefore a focus of this proposal. In specific aim #1, our innovative studies will utilize molecular and immunological tools to evaluate the contribution of TLR3 to V&#945;14iNKT cell activation during acute viral infections. In specific aim #2, we will examine the functional consequences of V&#945;14iNKT cell activation via TLR3 signaling on the innate immune response during acute viral infection. Replication-defective Adenoviruses are the most widely studied and used replication-defective vaccine vectors. Replication-defective Adenoviruses are important probes of the host immune response. They help define the first round of infected cells in host immune responses as well as the mechanism of activation of the innate immune response. Therefore, this virus is the obvious choice to use in our studies to evaluate the potential diversity in V&#945;14iNKT cell activation and function in the presence and absence of acute viral replication. Our preliminary studies have identified a novel and important biological role for TLR3 as a potential mediator of V&#945;14iNKT cell activation during acute viral infection. Specifically, in response to replication-defective Adenovirus infection in mice, TLR3 negatively regulates V&#945;14iNKT cell activation. In contrast, TLR3 positively regulates V&#945;14iNKT cell activation during replication-competent Adenovirus infection in mice. These preliminary findings provide support for a key part of our hypothesis that TLR3 differentially regulates V&#945;14iNKT cell activation in the presence and absence of acute viral replication to differentially alter the innate immune response. Overall, our studies will advance basic immunological knowledge by providing novel strategies/insights for manipulating V&#945;14iNKT cell activation. Valuable information obtained from our studies will enhance the development of replication-defective Adenovirus as vectors for gene therapy and vaccines. Moreover, these vaccines may one day be used for the prevention of devastating diseases by replication-competent viruses including HIV, Influenza A virus and herpes simplex virus.